Intragraft expression of transforming growth factor-beta 1 by a novel quantitative reverse transcription polymerase chain reaction elisa in long lasting kidney recipients

Background. Chronic allograft nephropathy (CAN) remains a major problem in clinical transplantation. It has been associated with increased transforming growth factor (TGF-β1). Our goal was to correlate CAN and levels of TGF-β1 by using a novel competitive quantitative for reverse transcription-polymerase chain reaction-ELISA (RT-PCR-ELISA) assay. Methods. We studied 12 transplantation patients (posttransplant time: 36.5±11.2 months, range (r): 13-52) with stable creatinine and blood pressure and varled proteinuria. A Kidney biopsy was performed in all patients. Six patients with acute tubular necrosis (ATN) immediately after transplantation were used as controls. Histopathological evaluation was based on Banff working classification criteria. We designed an heterologous RNA competitor (IC) for RT-PCR-ELISA, which co-amplified with the same primer as TGF-β1. Products were viewed on 96-well plates labeled with probes for IC at the desired sequence. Results. Results were expressed as the number of TGF-β1 copies/μg of total RNA. Six patients showed more than 1000 mg/24 hr proteinuria (2446±1421 mg/24 hr, r: 1200-5000) higher CAN Banff scores, and the other six presented <1,000 mg/24 hr (348±267 mg/24 hr, r: 114-800). This difference was significant (P=0.01). There were not significant differences in posttransplant time, creatinine, or blood pressure between groups. TGF-β1 levels by RT-PCR-ELISA were statistically significant (6038±5317, r: 1239-12100 versus 177±119.7, r: 51-400, P=0.04). The control group showed levels of 228±111, r: 140-444, P=0.04) with significant difference only for the higher proteinuria group (P=0.03). Conclusions. This study showed that those patients with elevated CAN scores and higher proteinuria levels had higher TGF-β1 intragraft expression.